Last year a new strategy was developed for the construction of a live attenuated influenza A virus vaccine in which attenuating mutations were introduced into a cDNA copy of the PB2 gene by site-directed mutagenesis and the mutant PB2 gene was subsequently rescued into an infectious virus. This year we have extended these initial findings considerably by demonstrating the feasibility of introducing two ts mutations into the PB2 gene that specify a greater degree of attenuation and temperature sensitivity than that specified by single a ts mutation. A double mutant was constructed that had ts mutations at amino acid positions 265 and 556 in the PB2 gene of the A/AA/6/60 virus. This double mutant was more temperature sensitive and more attenuated than a transfectant bearing a single mutation at position 265 or 556. Thus, in the near future it should be possible to construct a PB2 gene that specifies an increased level of attenuation that is very stable. Such a PB2 gene with multiple mutations could be used alone, or in conjunction with another attenuating gene, to attenuate new epidemic influenza A viruses as they emerge in nature. A mutation in the PA polymerase protein at amino acid residue 245 or 347 can suppress the ts and attenuation phenotypes specified by an asn to ser mutation at position 265 in the PB2 gene. Primary pulmonary cytotoxic CD8+ T-cells induced by immunization of mice with vaccinia recombinants expressing the major influenza A virus NP CTL epitope did not protect the mice against challenge with virulent virus.